Primer probe combination, kit, method and application for detecting methylation of DNAH17 gene

Abstract

The invention relates to a primer probe combination and a kit for detecting methylation of a DNAH17 gene as well as a method and application of the primer probe combination and the kit. According to the methylation principle, the specific DNAH17 gene methylation primer and probe are specifically designed, and the fluorescent quantitative PCR method is combined, so that the kit has the characteristics of high specificity, high sensitivity and the like, and is beneficial to assisting clinical diagnosis and developing related subsequent treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer-probe combination, a kit, a method and an application thereof for detecting DNA H17 gene methylation. Background Art

[0002] The DNAH17 (dyneinaxonemalheavychain17, DNAH17) gene is located on chromosome 17q25.3. It is a large gene composed of 81 exons. It encodes the axonemal heavy chain of ODA, which is the basic structure of primary cilia. In recent years, through genome sequencing, the DNAH17 gene has been found to be possibly associated with azoospermia, which is a common cause of male infertility, but its etiology is still not completely clear. The axonemal chain is the core structure of the sperm flagellum and runs through the entire length of the flagellum. Currently, the most studied are the DNAH17 gene-related SNP sites, and there are fewer studies on the effects of DNAH17 gene methylation on azoospermia. DNAH1 and DNAH9 genes are homologous genes of the DNAH17 gene, which have been proven to play an important role in the formation and regulation of cilia / flagella, and the loss of primary cilia has been found in many tumors and is gradually considered to be one of the tumor markers. In addition, studies have found that in liver cancer tissue, low methylation occurs in the DNAH17 promoter region, causing overexpression of DNAH17 protein, which may promote the occurrence and development of liver cancer, and is expected to become a biomarker with the presence of liver cancer thrombus. Therefore, the present invention aims to study the methylation of DNAH17 gene in semen, tissue or blood, whether it is consistent with the reported DNAH17 gene methylation trend. At present, there are many factors affecting azoospermia, and it is necessary to do more examinations (including imaging, slice tissue examination, etc.) to confirm the diagnosis, which takes a long time and the patient's waiting time is also long. The present invention is directed to the study of DNAH17 gene methylation, which can be used for early prediction of tumors, assisting clinicians to eliminate some pathogenic factors, and determine the correct treatment direction.

[0003] At present, there are many methods for detecting genomic DNA methylation, and the most common ones are fluorescent quantitative PCR, bisulfite treatment combined with sequencing, HRM technology, pyrophosphate sequencing, etc. The advantages of bisulfite treatment combined with sequencing are reliable results and high accuracy, which can clearly determine the methylation status of each CpG site in the target fragment, but the process is relatively cumbersome and time-consuming; HRM technology detects the methylation status of the target gene based on changes in melting temperature and peak shape, but this method has high requirements for instruments; pyrophosphate sequencing can detect and quantify subtle changes in methylation levels, but this method is more suitable for large sample size detection and has a high cost; while fluorescent quantitative PCR technology can be applied to the detection of a small number of samples, with high sensitivity and specificity, low cost compared to other methods, simple detection method, and strong operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0004] Figure 1 This is the result of multiplex fluorescence quantitative PCR amplification of DNAH17 gene methylation-positive samples.

[0005] Figure 2 This is the result of multiplex fluorescence quantitative PCR amplification of DNAH17 gene methylation negative samples.

[0006] Figure 3 This is the result of multiplex fluorescence quantitative PCR amplification of DNAH17 gene methylation invalid samples.

[0007] Figure 4 This is the sequencing result of the DNAH17 gene methylation-positive sample. The four CG sites at the probe position remain unchanged, so it is judged that the DNAH17 gene is fully methylated.

[0008] Figure 5 This is the sequencing result of the DNAH17 gene methylation-positive sample. The CG sites of probes 3 and 4 remain unchanged, and the CG sites of probes 1 and 2 are TG / CG. Therefore, it is judged that the DNAH17 gene is methylated and part of it is unmethylated.

[0009] Figure 6 This is the sequencing result of the DNAH17 gene methylation-negative sample. The four CG sites at the probe position changed to TG, so it was judged that the DNAH17 gene was not methylated. Summary of the invention

[0010] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a primer-probe combination for detecting DNAH17 gene methylation.

[0011] The present invention also provides an application of the primer-probe combination for methylation detection.

[0012] The present invention also provides a method for detecting DNAH17 gene methylation.

[0013] According to one aspect of the present invention, a primer-probe combination for detecting DNAH17 gene methylation is proposed, wherein the detection primer-probe combination comprises a pair of forward primers and reverse primers and a fluorescent probe, and the sequences are shown in SEQ ID NO:1 to SEQ ID NO:3.

[0014] Preferably, the fluorescent probe has a luminescent group FAM at the 5' end and a quenching group BHQ1 at the 3' end.

[0015] Preferably, the primer-probe combination for detecting DNAH17 gene methylation is only used to detect the sequence of DNAH17 gene modified by a sulfiding agent; the sulfiding agent refers to a reagent that converts unmethylated cytosine in DNA into uracil while leaving 5-MeC substantially unaffected.

[0016] Preferably, the sulfiding agent is bisulfite.

[0017] Preferably, the detection region of the DNAH17 gene targeted by the primer-probe combination for detecting DNAH17 gene methylation is close to the promoter region.

[0018] Preferably, the detection region targeted by the primer-probe combination for DNAH17 gene methylation detection is the CG-enriched region of the DNAH17 gene.

[0019] Preferably, the primer-probe combination for detecting DNAH17 gene methylation also contains a primer-probe combination for detecting an internal reference gene ACTB, the primer sequences containing the internal reference gene are shown in SEQ ID NO:4 to SEQ ID NO:5, and the probe sequence is shown in SEQ ID NO:6.

[0020] The second aspect of the present invention provides an application of the primer-probe combination for detecting DNAH17 gene methylation in preparing a product for detecting DNAH17 gene methylation. The product for detecting DNAH17 gene methylation includes a kit for detecting DNAH17 gene methylation.

[0021] The third aspect of the present invention provides a kit comprising the above-mentioned kit for detecting DNAH17 gene methylation.

[0022] Preferably, the kit contains the above-mentioned primer-probe combination for DNAH17 gene methylation detection, including a primer-probe combination of the internal reference gene ACTB.

[0023] Preferably, the working concentrations of the primer-probe combination for detecting methylation of the DNAH17 gene and the primer-probe combination for detecting the internal reference gene ACTB are both 10 uM.

[0024] Preferably, the kit also includes a multiplex fluorescence quantitative PCR reaction solution amplification system.

[0025] Preferably, the multiplex fluorescence quantitative PCR reaction solution amplification system further includes enzyme-free sterile water and 2×T5FastqPCR Mix (the final concentration in the final reaction system is 1×).

[0026] A fourth aspect of the present invention provides a method for detecting DNAH17 gene methylation, comprising: (1) extracting a sample DNA to be tested;

[0027] (2) treating the sample to be tested with sulfite to obtain a modified sample to be tested;

[0028] (3) Purifying the modified DNA sample to obtain purified DNA sample; (4) Using the kit to perform multiplex fluorescence quantitative PCR amplification on the DNA sample in step (3) to detect the methylation status of the DNA H17 gene.

[0029] The beneficial effects of the present invention include:

[0030] (1) The qPCR method is simple, fast, low-cost, high in specificity and sensitivity, and short in detection time, making it more suitable for clinical testing;

[0031] (2) The amplified fragment region of the present invention is close to the promoter CG-rich region before the CDS region (methylation generally occurs in the promoter region);

[0032] (3) According to the methylation principle: after DNA is modified by bisulfite, the methylated cytosine remains unchanged, the unmethylated cytosine is converted into uracil, and the DNA gene sequence remains unchanged. The specific primers and probes designed by the present invention are only targeted at the DNA sequence after bisulfite modification, and have strong specificity;

[0033] (4) Based on this point in (2), when designing primers, the upstream primer introduces a degenerate base Y, and the downstream primer does not have a CG site, so a degenerate base is not required. This is because CG has three possibilities after sulfurization: fully methylated CG, partially methylated CG / TG, and completely unmethylated TG. The upstream and downstream primers designed according to the present invention can amplify all these situations;

[0034] (5) The probe sequence is covered with four methylated CG sites, which are close to the 3' end of the sequence to ensure amplification specificity; in addition, the DNAH17 gene probe sequence uses a fluorescent reporter group different from the internal reference gene ACTB probe;

[0035] (6) The internal reference gene ACTB and the target gene DNAH17 can be detected simultaneously in the same tube, which can reduce the risk of contamination of the sample to be tested and reduce the cost of the amplification primer probe combination reagent. The amplification results are the same as those of separate detection.

[0036] Example 1

[0037] Design of primer and probe combination for DNAH17 gene methylation detection

[0038] The design process of the primer probe combination for DNAH17 gene methylation detection is as follows: find the genomic sequence of the DNAH17 gene in the database, with reference to the sequence NC_000017.11 (partial sequence is shown in SEQ ID NO: 7):

[0039] 1) Methylation sequence: All Cs other than CpG are converted to T to form a methylation sequence, and the sequence is shown in SEQ ID NO: 8. The CpG islands are analyzed, and the appropriate regions are obtained and primers and probes are designed using PRIMER5 software. The designed primers and probes are primers and probes used for multiplex fluorescence quantitative PCR amplification to detect the methylation of the DNAH17 gene, wherein the internal reference gene is ACTB, and the methylation sequence of the internal reference gene is shown in SEQ ID NO: 9.

[0040] 2) Unmethylated sequence: that is, the sequence of DNAH17 gene that is not methylated (DNAH17 gene methylation negative) as shown in SEQ ID NO:10.

[0041] Primer probe design specifically includes the following:

[0042] 1. Selection of detection area

[0043] Since the methylation status and distribution of the same gene are not uniform, the results of selecting different regions for amplification may also be different. The amplified region selected in the present invention is the CG-rich region near the promoter before the CDS region of the DNAH17 gene, and its original sequence is shown in SEQ ID NO:7.

[0044] 2. Design a pair of methylation primers and a probe for the CG-rich region of the original sequence

[0045] According to the methylation principle: all Cs other than CpG are converted into Ts, and the original DNAH17 fragment is converted into a methylated fragment, whose sequence is shown in SEQ ID NO:8. A forward primer DNAH17-QP-F, a reverse primer DNAH17-QP-R and a probe DNAH17-QP-P are designed on the methylated fragment of the DNAH17 gene, and their sequences are shown in SEQ ID NO.1 to SEQ ID NO.3, wherein the fluorescent probe DNAH17-QP-P has a luminescent group FAM at the 5' end and a quenching group BHQ1 at the 3' end; wherein the forward primer DNAH17-QP-F introduces a degenerate base Y, and can amplify both the methylated sequence (sequence as shown in SEQ ID NO:8) and the non-methylated sequence (sequence as shown in SEQ ID NO:10).

[0046] In addition, the forward primer ACTB-F, reverse primer ACTB-R and probe ACTB-P designed for the methylation sequence of the internal reference gene ACTB (sequence as shown in SEQ ID NO:9) have sequences as shown in SEQ ID NO.4 to SEQ ID NO.6, wherein the fluorescent probe ACTB-P has a luminescent group HEX at the 5' end and a quenching group BHQ1 at the 3' end.

[0047] The relevant sequence information involved in Example 1 is shown in Table 1.

[0048] Table 1. Sequence information

[0049]

[0050]

[0051] Example 2

[0052] This embodiment prepares a multiplex fluorescence quantitative PCR amplification kit for DNAH17 gene methylation detection, including the primer probe set DNAH17-QP-F, DNAH17-QP-R and DNAH17-QP-P prepared in Example 1, the primer probe set ACTB-F, ACTB-R and ACTB-P of the internal reference gene, PCR reaction solution, positive control and negative control, and the working concentration of the primer probe combination is 10uM. The PCR reaction solution also includes: enzyme-free sterile water, and the final concentration of 2×T5 Fast qPCR Mix (Probe) in the final reaction system is 1× (purchased from Qingke Biology).

[0053] Example 3

[0054] Method for detecting DNAH17 gene methylation

[0055] 1. Extract the DNA of the semen sample to be tested according to the blood genomic DNA extraction kit (DP348, TIANGEN)

[0056] 1.1 First, homogenize the sample, then pipette 200ul of the sample into a 1.5ml EP tube.

[0057] 1.2 Add 20ul proteinase K solution and mix well.

[0058] 1.3 Add 200ul of buffer GB, mix thoroughly by inversion, and place at 56℃ for 10 minutes, inverting several times to mix, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0059] 1.4 Add 200ul of anhydrous ethanol, mix thoroughly by inversion, and centrifuge briefly to remove water droplets on the inner wall of the tube cap.

[0060] 1.5 Add the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000rpm (13400×g) for 30 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CB3 back into the collection tube.

[0061] 1.6 Add 500ul of buffer GD to the adsorption column CB3, centrifuge at 12000rpm (13400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0062] 1.7 Add 600ul of rinse solution PW to the adsorption column CB3, centrifuge at 12000rpm (13400×g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in a collection tube.

[0063] 1.8 Repeat step 1.7 once.

[0064] 1.9 Centrifuge at 12000rpm (13400×g) for 2 minutes and discard the waste liquid. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse liquid in the adsorption material.

[0065] 1.10 Transfer the adsorption column CB3 into a clean centrifuge tube, add 50ul of elution buffer TB to the middle part of the adsorption membrane, let it stand at room temperature for 2-5 minutes, centrifuge at 12000rpm (13400×g) for 2 minutes, and collect the solution into the centrifuge tube.

[0066] 1.11 Use Nano Drop 2000 to measure the concentration of the extracted DNA. If you do not continue the next experiment, store it at -20℃.

[0067] 2. Sulfurization conversion

[0068] 2.1 Prepare bisulfite solution according to the instructions of the Methylation Detection Pretreatment Kit (Magnetic Bead Method) (careLYFE) kit. Add 950ul of buffer BML to each tube of bisulfite dry powder and shake for 5 minutes to ensure that the dry powder is completely dissolved.

[0069] 2.2 Prepare bisulfite reaction, the formula is shown in Table 2:

[0070] Table 2. Bisulfite reaction system

[0071] Components Dosage DNA samples 40ul Buffer DPL 10ul Bisulfite solution 100ul Total volume 150ul

[0072] 2.3 Perform bisulfite sulfidation reaction on a PCR instrument. The procedure is shown in Table 3:

[0073] Table 3. Bisulfite sulfurization reaction conditions

[0074] temperature time 95℃ 5 minutes 64℃ 3 hours 4℃ No more than 20 hours

[0075] 3. Purify DNA using the methylation detection pretreatment kit (magnetic bead method) (careLYFE) 3.1 After the bisulfite treatment, transfer the system in the reaction tube to a clean 1.5 ml centrifuge tube after a brief centrifugation.

[0076] 3.2 Add 1 ml of buffer GDF and 40 ul of magnetic bead suspension E, mix for 10 seconds, and let stand at room temperature for 5 minutes. 3.3 Place the centrifuge tube in a magnetic field for 1 minute, slowly aspirate the liquid and discard it.

[0077] 3.4 Remove the centrifuge tube from the magnetic field, add 600ul of buffer PWG and mix for 10 seconds.

[0078] 3.5 Place the centrifuge tube in a magnetic field and let it stand for 1 minute. Slowly aspirate the liquid and discard it.

[0079] 3.6 Remove the centrifuge tube from the magnetic field, add 600ul of buffer DB and mix for 10 seconds, then let stand at room temperature for 15 minutes.

[0080] 3.7 Place the centrifuge tube in a magnetic field and let it stand for 1 minute. Slowly aspirate the liquid and discard it.

[0081] 3.8 Remove the centrifuge tube from the magnetic field, add 600ul of buffer PWG and mix for 10 seconds.

[0082] 3.9 Place the centrifuge tube in a magnetic field and let it stand for 1 minute. Slowly aspirate the liquid and discard it.

[0083] 3.10 Repeat steps 3.8 to 3.9.

[0084] 3.11 Briefly centrifuge to collect the remaining liquid at the bottom of the tube, and try to remove as much liquid as possible; dry at room temperature for 5 minutes until there is no liquid left and the magnetic beads are not cracked.

[0085] 3.12 Add 40ul of elution buffer TBC and mix the magnetic beads by blowing and pipetting. Place the magnetic beads in a magnetic field. Wait for 3 minutes at room temperature, then aspirate the liquid into a new 1.5mL centrifuge tube to obtain the template for subsequent multiplex fluorescence quantitative PCR detection in the same tube.

[0086] 4. Multiplex fluorescence quantitative PCR detection

[0087] 4.1 Reagent preparation: Multiplex fluorescence quantitative PCR reaction reagents and DNAH17 gene and internal reference gene methylation primer and probe mixture. Prepare the reaction solution according to the following system based on the number of samples.

[0088] Table 4. Fluorescence quantitative PCR amplification system

[0089]

[0090] 4.2 Each experiment requires one negative control and one positive control.

[0091] 4.3 Aliquot 23ul of each tube into 0.2ml PCR reaction tubes.

[0092] 4.4 Add 2ul of the purified sample DNA after sulfurization treatment in step 3 above to the aliquoted PCR reaction tube, and the total volume is 25ul.

[0093] 4.5 Perform multiplex fluorescence quantitative PCR reaction, and use the two-step fluorescence quantitative PCR amplification standard procedure as follows:

[0094] Table 5. Fluorescence quantitative PCR amplification program

[0095]

[0096] 5. Results Analysis

[0097] After the reaction is completed, the results are automatically saved, and the instrument supporting software is used to automatically analyze the results. The Start value, End value, and Threshold value of the Baseline are adjusted according to the analyzed image (the user can adjust them according to the actual situation. The instrument used in the present invention is ABI7500, the Start value is 3, the End value is 15, and the fluorescence threshold (Threshold) is set to 0.01), and click Analysis to automatically obtain the analysis results.

[0098] 6. Result determination

[0099] The interpretation of the multiplex fluorescence quantitative PCR reaction results is shown in Table 6:

[0100] When the internal reference gene ACTB is positive, the DNAH17 gene methylation test result is positive / negative and the result is considered valid;

[0101] When the internal reference gene ACTB is negative, the result of DNAH17 gene methylation test is considered invalid, and the sample DNA will be re-extracted for testing.

[0102] Criteria for positive judgment of target gene DNAH17 methylation: If the result is valid, a Ct value > 38 is negative, and a Ct value ≤ 36 is positive. Recheck if the Ct value is between 36-38;

[0103] Table 6. Interpretation of individual PCR reaction results

[0104]

[0105] Example 4

[0106] Clinical sample testing

[0107] Ten clinical samples were selected, and genomic DNA was extracted and purified by sulfurization according to the method described in Example 3, and reagents were prepared and multiplex fluorescence quantitative PCR was performed. The amplification results are shown in Figure 1 , 2 and 3: DNAH17 gene methylation amplification positive samples are shown in only one example. Figure 1 As shown: both the reference gene ACTB and the target gene DNAH17 were typically amplified and the Ct value was ≤36; the negative result of DNAH17 gene methylation amplification was as shown Figure 2 As shown: only the internal reference gene ACTB has an amplification curve Ct value ≤ 36, and the target gene DNAH17 has no amplification curve; the DNAH17 gene methylation amplification invalid result is as follows Figure 3 As shown: only the internal reference gene ACTB has an amplification curve but the Ct value is > 36. The Ct value results after multiplex fluorescence quantitative PCR detection are shown in Table 7: The methylation results of the DNAH17 gene were determined according to Example 3.

[0108] Table 7. Results of multiplex fluorescence quantitative PCR amplification of 10 samples

[0109]

[0110] Undetermined means that no valid value can be detected under the threshold set by this program. Generally, if Ct>=40, the real result is not displayed but undetermined is used instead.

[0111] Example 5

[0112] The DNAH17 gene methylation positive samples 28, 22 and negative sample W-900 in Example 4 were sequenced as examples to verify the accuracy of the primers and methods designed in Example 1. After PCR amplification and sequencing, the results were as follows Figure 4 , 5 and 6 shown: by Figure 4 The sample 28 with positive DNAH17 gene methylation was shown. The sequencing sequence of the CG position of the methylation sequence of this sample was CG, indicating that the CG position of the DNA sequence remained unchanged after bisulfite sulfidation and complete methylation occurred. The result was consistent with the method developed by the present invention ( Figure 4 The probe sequence in the figure is reverse sequence, so it is numbered from right to left); Figure 5 The sample 22 with positive DNAH17 gene methylation was shown. The sequencing sequence of the CG position of the methylation sequence of this sample was CG / TG, indicating that after the DNA sequence was treated with bisulfite, the sample was methylated, but part of it was unmethylated. Figure 6The sample W-900 with negative DNA H17 gene methylation showed that the sequencing sequence of the CG position of the methylation sequence of this sample was TG, indicating that the DNA sequence was not methylated after bisulfite sulfonation, and the result was consistent with the method developed by the present invention. Therefore, it is shown that the primer probe, kit and detection method of the present invention can be used for sample DNA H17 gene methylation detection, and the result is accurate.

Claims

1. A primer-probe combination for detecting DNAH17 gene methylation, characterized in that: The primer-probe combination comprises a pair of forward primers, a reverse primer and a fluorescent probe, wherein the sequences of the pair of forward and reverse primers are shown in SEQ ID NO:1-SEQ ID NO:2, and the sequence of the fluorescent probe is shown in SEQ ID NO:

3. The fluorescent probe has a luminescent group FAM at its 5' end and a quenching group BHQ1 at its 3' end.

2. The primer-probe combination for detecting DNAH17 gene methylation according to claim 1, characterized in that: It also includes a pair of forward primers and reverse primers of the internal reference gene ACTB and a fluorescent probe, whose sequences are shown in SEQ ID NO:4-SEQ ID NO:

6. The fluorescent probe has a luminescent group HEX at the 5' end and a quenching group BHQ1 at the 3' end.

3. The primer-probe combination for detecting DNAH17 gene methylation according to claim 1, characterized in that: The primer-probe combination for detecting DNAH17 gene methylation is only used to detect the sequence of DNAH17 gene modified by a sulfiding agent, and the sulfiding agent is bisulfite.

4. The primer-probe combination for detecting DNAH17 gene methylation according to claim 1, characterized in that: The detection region of the DNAH17 gene by the primer-probe combination is near the promoter region of the DNAH17 gene, and the original sequence of the detection region is shown in SEQ ID NO:

7. The methylation detection region sequence of the original sequence after bisulfite treatment is shown in SEQ ID NO:

8.

5. The primer-probe combination for detecting DNAH17 gene methylation according to claim 1, characterized in that: The primer probe set also includes detection of the methylation region of the internal reference gene ACTB, and the sequence of the methylation detection region of the internal reference gene ACTB is shown in SEQ ID NO:

9.

6. Use of the primer-probe combination for detecting DNAH17 gene methylation according to claim 1 in preparing a product for detecting DNAH17 gene methylation.

7. A kit for detecting DNAH17 gene methylation, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 5.

8. A method for detecting DNA H17 gene methylation for non-disease diagnosis purposes, characterized in that: The following steps are involved: (1) Extracting DNA from the sample to be tested; (2) treating the sample to be tested with bisulfite to obtain modified DNA of the sample to be tested; (3) purifying the modified DNA sample to obtain purified DNA sample; (4) Using the DNAH17 gene methylation detection primer probe combination described in any one of claims 1 to 7, the DNAH17 gene methylation status of the sample to be tested in step (3) is detected.

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